The objective of this project is to define the initial, intra-cellular events of steroid hormone action. Synthetic glucocorticoid derivatives, some of which react to form covalently labeled receptors via affinity labeling, have been prepared and are being used with glucocorticoid-responsive rat hepatoma tissue culture cells to examine: (1) steroid-receptor binding site interactions; (2) the effects of steroid binding on receptor conformation; and (3) the nature of "activation" of receptor-steroid complexes. We have combined affinity labeling by dexamethasone 21-mesylate with protease digestion in order to study various physical states (i.e., activated and unactivated) of covalent receptor-steroid complexes and to assess receptor-homology in different target tissues. These studies indicate that if activation of glucocorticoid receptor-steroid complexes is accompanied by changes in conformation or oligomerization (as has suggested by others), the changes are not detectable by these methods and must be of limited magnitude. We have also synthesized a highly fluorescent glucocorticoid derivative (Dex-C2-Rho). This steroid yielded the first specific, fluorescent glucocorticoid receptor-steroid complex, which should be very useful in the study of receptor-steroid complexes by fluorescence spectroscopy.